Integrated Computational Element Analytical Methods for Microorganisms Treated with a Pulsed Light Source

ABSTRACT

Determining the microorganism load of a substance may be conducted readily using one or more integrated computational elements. By determining a substance&#39;s microorganism load, the substance&#39;s suitability for a variety of applications may be ascertained. Methods for determining the microorganism load of a substance using one or more integrated computational elements can comprise: providing a substance comprising a plurality of viable microorganisms; exposing the substance to a pulsed light source for a sufficient length of time to form at least some non-viable microorganisms; and determining a microorganism load of the substance using one or more integrated computational elements.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 13/204,294, filed on Aug. 5, 2011, which is incorporated hereinby reference in its entirety.

BACKGROUND

The present invention generally relates to the monitoring ofmicroorganisms, and, more specifically, to the use of one or moreintegrated computational elements to determine the effectiveness of amicroorganism treatment.

The presence of bacteria and other microorganisms in a substance isoften determined after enhancing low levels of biological material todetectable levels. In some cases, an aliquot of the substance can becultured under conditions that are conducive for growth of a particularbiological material. In other cases, nucleic acid amplificationtechniques, such as polymerase chain reaction (PCR), can be used toincrease levels of nucleic acids. Culturing methods, in particular, maysometimes be non-specific, as many different types of microorganisms maygrow under the chosen culturing conditions, whereas only certainmicroorganisms may be of interest for an analysis. Furthermore, bothculturing and nucleic acid amplification techniques are oftenconstrained by the timeframe over which they are conducted. PCRtechniques, for example, may take several hours or more to producesufficient nucleic acid quantities for analysis, and culturing may takedays to weeks to complete. Methods for real-time or near real-timemonitoring of bacteria and other microorganisms are not believed to haveyet been developed.

The present inability to monitor bacteria and other microorganisms in asufficiently rapid manner can have significant ramifications for avariety of commercial and industrial products and processes. Forexample, due to a limited shelf life, a product (e.g., a foodstuff orpharmaceutical) may have been transported to a store and released forpublic consumption before product quality testing has been fullycompleted. By the time a biological contamination has been uncovered, itcan oftentimes be too late, as consumers may have already come incontact with the contaminated product. Not only can human health becompromised, but valuable process time, raw materials, and otherresources may have been lost by preparing and distributing acontaminated product.

Although biological contamination is a recognizable concern in the foodand drug industry, the problem of contamination by bacteria and othermicroorganisms extends to a much broader array of fields, includingthose not directly impacting human health. For example and withoutlimitation, biological monitoring of water treatment and wastewaterprocessing streams, including those from refineries, can be ofsignificant interest due to downstream contamination issues. Insubterranean operations, biological contamination can reduce productionand/or result in biofouling of equipment and wellbore surfaces. Inaddition, biological contamination on some solid surfaces can lead tostructural defects, including corrosion, that ultimately may result inmechanical failure. In short, any industry in which monitoring ofbiological contamination is of interest could potentially benefit frommore rapid detection techniques for biological materials.

Concurrently with monitoring for the presence of biological materials,there often can be an interest in reducing and/or preventing biologicalcontamination within a substance, including on a surface. In someinstances, a biocide may be used to slow or stop biological growth.Although biocides may often be effective for addressing biologicalcontamination, their effects can sometimes be slow acting. In addition,at least some members of a population of microorganisms usually survivetreatment with a biocide. Another technique that may be used to slow orstop biological growth is irradiation with a source of electromagneticradiation (e.g., ultraviolet radiation). Continuous-operationultraviolet radiation sources, particularly mercury vapor ultravioletradiation sources, are often used for this purpose.

Some bacteria and other microorganisms, instead of being killed outrightby continuous-operation ultraviolet radiation sources, may undergo atransformation whereby they are still metabolically active but are nolonger able to reproduce. Without being bound by any theory ormechanism, it is believed that the microorganisms, when transformed,contain nucleic acid damage that renders them incapable of reproducingbut still having an intact cell wall that allows them to remaintemporarily viable. In many instances, these altered microorganisms canbe externally indistinguishable from unaltered microorganisms, therebymaking it difficult to determine how effectively a biologicalcontamination has been addressed until time-consuming culturing testshave been completed. In addition, while still living, the alteredmicroorganisms can still cause detrimental effects, including thosenoted above.

SUMMARY OF THE INVENTION

The present invention generally relates to the monitoring ofmicroorganisms, and, more specifically, to the use of one or moreintegrated computational elements to determine the effectiveness of amicroorganism treatment.

In some embodiments, the present invention provides a method comprising:providing a substance comprising a plurality of viable microorganisms;exposing the substance to a light source for a sufficient length of timeto form at least some non-viable microorganisms; and determining amicroorganism load of the substance using one or more integratedcomputational elements.

In some embodiments, the present invention provides a method comprising:measuring viable microorganisms in a substance, identifying one or moretypes of microorganisms in a substance, or any combination thereof usingone or more integrated computational elements; after measuring viablemicroorganisms or identifying one or more types of microorganisms in thesubstance, exposing the substance to a pulsed light source operable forrendering at least a portion of the microorganisms non-viable; and afteror while exposing the substance to the pulsed light source, determininga microorganism load of the substance using one or more integratedcomputational elements.

In some embodiments, the present invention provides a device comprising:a pulsed light source configured to expose a substance toelectromagnetic radiation suitable for rendering one or moremicroorganisms non-viable; and one or more integrated computationalelements configured for determining a microorganism load of thesubstance after or during its exposure to the pulsed light source.

The features and advantages of the present invention will be readilyapparent to one having ordinary skill in the art upon a reading of thedescription of the preferred embodiments that follows.

BRIEF DESCRIPTION OF THE DRAWING

The following FIGURE is included to illustrate certain aspects of thepresent invention, and should not be viewed as an exclusive embodiment.The subject matter disclosed is capable of considerable modification,alteration, and equivalents in form and function, as will occur to onehaving ordinary skill in the art and the benefit of this disclosure.

FIG. 1 shows a schematic of an illustrative integrated computationalelement (ICE).

DETAILED DESCRIPTION

The present invention generally relates to the monitoring ofmicroorganisms, and, more specifically, to the use of one or moreintegrated computational elements to determine the effectiveness of amicroorganism treatment.

As discussed above, conventional methods for monitoring and addressingbiological contamination may be limited both by their effectiveness andtimeliness of producing results. In order to address the foregoingissues and others, devices and methods described herein have beendeveloped that may enhance the effectiveness of remediating biologicalcontamination and enable rapid determination of a treatment'seffectiveness. In particular, the devices and methods described hereinutilize a pulsed light source in combination with one or more integratedcomputational elements to accomplish the foregoing. The pulsed lightsource may result in a more effective remediation of microorganisms thando continuous-operation (i.e., non-pulsed) light sources, as discussedhereinafter. In addition, detection and analysis of microorganisms usingone or more integrated computational elements may take place much morerapidly than through conventional biological assays, such as culturingand PCR.

The methods and devices described herein may be used in any field whereit is desirable to assay for microorganisms and/or determine theeffectiveness of a remediation operation used to control microorganisms.Given the benefit of the present disclosure, one having ordinary skillin the art will be able to apply the techniques described herein to anyapplication in which it is desirable to control and measuremicroorganisms in a substance. Without limitation, the methods anddevices described herein may be used in fields such as, for example;water analyses, including drinking water, waste water, and processingwater analyses; foodstuff, beverage, pharmaceutical, and cosmeticanalyses; surface analyses; oil, gas, treatment fluid, drilling mud, andsubterranean fluid analyses; and the like. In addition, the methods anddevices described herein may be used in the healthcare industry to assayfor biological contamination on surfaces such as, for example medicaldevices, surgical instruments, and the like. Other industries where itmay be desirable to monitor for biological contamination on a surfacemay be envisioned by one having ordinary skill in the art.

In contrast to continuous-operation light sources, pulsed light sourcesdeliver short bursts of highly intense electromagnetic radiation to asubstance to address biological contamination therein. It is believedthat pulsed electromagnetic radiation is often much more damaging tobiological materials than is non-pulsed electromagnetic radiation andmay result in more effective biological remediation of a substance.Without being bound by any theory or mechanism, it is believed thatpulsed light sources, in contrast to continuous-operation light sources,may reduce the integrity of microorganisms' cell walls, therebyresulting in significantly increased outright killing of themicroorganisms to render them non-viable. By achieving outright killingof the microorganisms, one may eliminate the possibility of themicroorganism being able to cause further issues while still remainingviable, such as illness, turbidity, and biofouling, for example.

As disclosed in commonly owned U.S. patent application Ser. No.13/204,294, filed on Aug. 5, 2011 and incorporated herein by referencein its entirety, one or more integrated computational elements may beused to rapidly detect and analyze particular types of bacteria,including whether the bacteria are living or dead. Those techniques maybe extended to other types of microorganisms, as discussed hereinafter.Microorganism analyses may be conducted using one or more integratedcomputational elements much more rapidly than with conventionalbiological assays. The rapidity by which integrated computationalelements may perform analyses is advantageous for a number ofapplications, and it is particularly advantageous for analyses ofbiological materials, including microorganisms.

The rapid analyses offered by integrated computational elements may beparticularly advantageous when used to analyze bioremediation that hasbeen conducted using a pulsed light source. Specifically, the integratedcomputational elements may be used to assess the degree to whichmicroorganisms or classes of microorganisms have been renderednon-viable by a pulsed light treatment. As noted above, use of a pulsedlight source may result in significantly increased outright killing ofmicroorganisms through cell wall integrity disruption relative toinactivation through nucleic acid damage, although the present methodsare not limited to these or other mechanisms of action. Differentiationbetween viable microorganisms and inactivated microorganisms may bedifficult to detect, and it may be problematic to determine theeffectiveness of a biological remediation until culturing or a relatedtechnique has taken place to determine viability. However,differentiation between viable microorganisms and non-viablemicroorganisms that have been killed outright through cell wallmodifications may be determined readily using one or more integratedcomputational elements, as described herein. For example, in someembodiments, the foregoing may be accomplished by using an integratedcomputational element that is configured for detecting the originalviable microorganisms and an integrated computational element that isconfigured for detecting non-viable microorganisms that have beenaltered by altering their cell walls. To measure an amount ofmicroorganisms in the substance, an output of the integratedcomputational element may be correlated with a concentration of themicroorganisms in a substance.

Due to the rapidity at which integrated computational elements mayprovide information about a population of microorganisms, they may beused advantageously for conducting real-time or near real-timebiological analyses, thereby satisfying an unmet need in the art.Furthermore, they may be used to follow and proactively manage theprogress of a biological remediation operation (e.g., using a pulsedlight source) in real-time or near-real time. That is, feedback fromanalyses conducted using one or more integrated computational elementsmay be used to alter a biological remediation operation, particularlyone conducted using a pulsed light source, in order to improve itseffectiveness. For example, if an analysis indicates that unacceptablyhigh levels of viable microorganisms remain in a substance during orfollowing its exposure to a pulsed light source, the operationalparameters associated with the pulsed light source may be altered in anattempt to increase the treatment effectiveness (e.g., different pulselengths, pulse intensities, pulse sequences, pulse waveforms, number ofpulses, total exposure time, combinations thereof, and the like), or adifferent pulsed light source may be used if a particular one is notproducing a desired effect. Thus, the combination of a pulsed lightsource and one or more integrated computational elements for treatmentfeedback may be used to more effectively conduct biological remediationsof a substance. Conventional microorganism assay techniques, incontrast, are simply too slow to allow proactive management ofbiological remediation operations to take place.

As used herein, the term “electromagnetic radiation” refers to radiowaves, microwave radiation, infrared and near-infrared radiation,visible light, ultraviolet radiation, X-ray radiation and gamma rayradiation.

As used herein, the term “light source” refers to a device that emitselectromagnetic radiation. Thus, as used herein, light sources are notlimited to devices that only emit visible light. In some embodiments,light sources may be monochromatic, emitting substantially only a singlewavelength. In other embodiments, light sources may be polychromatic,emitting a plurality of wavelengths, which may comprise a range ofwavelengths.

As used herein, the term “continuous-operation light source” refers to alight source that continually produces electromagnetic radiation ofsubstantially the same output intensity.

As used herein, the term “pulsed light source” refers to a light sourcethat produces electromagnetic radiation not having the same outputintensity at all times. In some embodiments, a pulsed light source maybe cycled between a high intensity ON state and an OFF state. In some orother embodiments, a pulsed light source may be cycled between a highintensity first state and a low intensity second state, where the lowintensity state does not represent a state where the pulsed light sourceis completely turned OFF.

As used herein, the term “microorganism” refers to a unicellular ormulti-cellular microscopic life form. Microorganisms may include, butare not limited to, bacteria, protobacteria, protozoa, phytoplankton,viruses, fungi, and alga. It is to be recognized that somemicroorganisms may be large enough to be seen with the naked eye.

As used herein, the term “viable microorganisms” refers tomicroorganisms that are substantially unaltered from their native stateand are capable of normal metabolic activity, including reproduction. Asused herein, the term “non-viable microorganisms” refers tomicroorganisms that are no longer metabolically active. In someembodiments, non-viable microorganisms may refer to microorganisms thathave had their cell wall ruptured, degraded, or modified by exposure toa degradative agent, such as a pulsed light source, for example. As usedherein, the term “inactivated microorganisms” refers to microorganismsthat have been altered from their native state and are no longer capableof reproducing. The alteration to form inactivated microorganisms may betemporary or permanent. Permanent alterations may include nucleic acidmutations, for example. Temporary alterations may include, for example,environmental conditions (e.g., temperature or lack of an appropriatenutrient source) that impact the microorganism's ability to reproduce orotherwise perform normal metabolic functions, but from which themicroorganism may recover once returned to more favorable conditions. Inthe case of viruses, the term “viable viruses” refers to viruses thatare capable of infecting host cells and replicating therein, and theterm “non-viable viruses” refers to viruses that are incapable ofreplicating in host cells.

As used herein, the term “microorganism load” refers to the type and/orquantity of viable microorganisms and/or non-viable microorganisms in asubstance.

As used herein, the term “substance” and variations thereof refer to anyfluid or any solid substance or material. Solid substances or materialsmay include, but are not limited to, rock formations, concrete, metal,plastic, and the like.

As used herein, the term “fluid” refers to any substance that is capableof flowing, including particulate solids, liquids, gases, slurries,emulsions, powders, muds, glasses, any combination thereof, and thelike. In some embodiments, the fluid can comprise an aqueous fluid,including water, mixtures of water and water-miscible fluids, and thelike. In some or other embodiments, the fluid can comprise an oleaginousfluid or like hydrocarbon-based fluid.

As used herein, the terms “real-time” and “near real-time” refer to ananalysis of a substance that takes place in substantially the same timeframe as the interrogation of the substance with electromagneticradiation.

Without being bound by any particular theory or mechanism, it isbelieved that analyses of viable microorganisms and non-viablemicroorganisms in a substance may be based upon monitoring of internalstructures within the microorganisms. As discussed above, irradiatingmicroorganisms with a high intensity pulsed light source may result inincreased cell wall integrity disruption, thereby exposing themicroorganisms' internal structures (e.g., nucleus, ribosomes,endoplasmic reticulum, and the like). Once exposed, the internalstructures may be in a significantly different chemical environment thatcan be detected using one or more integrated computational elements. Insome embodiments, a dye or like tracer that interacts with the exposedinternal structures may be used to further enhance their detectability.Specifically, when low quantities of the internal structures are presentor they are only weakly spectrally active, one or more integratedcomputational elements configured to detect a complex between a dye orlike tracer and an internal structure may be used. In some or otherembodiments, detection of viable microorganisms and non-viablemicroorganisms may be based upon detection of the changes that occur intheir cell walls after being exposed to a pulsed light source. Whendetecting viable microorganisms and non-viable microorganisms by cellwall alterations, one or more integrated computational elements may beused that are configured to detect and analyze unaltered and alteredcell walls.

As discussed briefly above, it is believed that the types ofmicroorganisms applicable for analysis by the present methods are notparticularly limited. Again without being bound by any theory ormechanism, it is believed that determination of the remediationeffectiveness for a particular type of microorganism following itsexposure to a pulsed light source may be based upon morphologicalchanges that can be detected and quantified using one or more integratedcomputational elements. Detection of the changes induced inmicroorganisms following their exposure to a pulsed light source may bebased upon any of the techniques described above, for example. Since themorphological changes that occur following exposure to a pulsed lightsource may be substantially similar across various microorganism types,it is believed that any type of microorganism may be analyzed by themethods described herein. In various embodiments, the microorganismsbeing detected may comprise at least one type of microorganism selectedfrom the group consisting of bacteria (including aerobic bacteria andanerobic bacteria), protobacteria, protozoa, phytoplankton, viruses,fungi, alga, and any combination thereof. Particular classes of bacteriathat may be of interest include, for example, gram-positive andgram-negative bacteria, aerobic and anaerobic bacteria, sulfate-reducingbacteria, nitrate-reducing bacteria, or any combination thereof. In someembodiments, bacteria of genera such as, for example, Y-proteobacteria,α-proteobacteria, δ-proteobacteria, Clostridia, Methanohalophilus,Methanoplanus, Methanolobus, Methanocalculus, Methanosarcinaceae,Halanaerobium, Desulfobacter, Marinobacter, Halothiobacillus, andFusibacter may be detected and analyzed by the techniques describedherein. In more specific embodiments, bacteria of interest in theoilfield industry that may be detected and analyzed using the methodsdescribed herein include, for example, Desulfovibrio desulfuricans,Desulfovibrio vulgaris, Desulfosarcina variabilis, Desulfobacterhydrogenophilus, Bdellovibrio bacteriovorus, Myxococcus xanthus,Bacillus subtilis, and Methanococcus vannielii.

In some embodiments, methods described herein can comprise: providing asubstance comprising a plurality of viable microorganisms; exposing thesubstance to a light source for a sufficient length of time to form atleast some non-viable microorganisms; and determining a microorganismload of the substance using one or more integrated computationalelements. During the length of time they are being exposed, themicroorganisms may absorb a sufficient amount of electromagneticradiation to render them non-viable. In some embodiments, themicroorganism load may be determined after or while exposing thesubstance to the light source. In some embodiments, the microorganismload may be determined before exposing the substance to the lightsource. In still other embodiments, the microorganism load may bedetermined both before and after exposing the substance to the lightsource.

In some embodiments, the light source may comprise a pulsed lightsource. In some embodiments, the pulsed light source may comprise apulsed ultraviolet (UV) light source. In some embodiments, the lightsource may comprise a continuous-operation light source, such as amercury vapor UV light source, for example. In still other embodiments,the light source may comprise a combination of a continuous-operationlight source and a pulsed light source. For example, in someembodiments, a pulsed UV light source may be used in combination with acontinuous-operation mercury vapor UV light source. Use of thecombination of a continuous-operation light source and a pulsed lightsource may be advantageous for producing microorganism activation bymultiple mechanisms (e.g., via genetic damage and cell wall integritydisruption). When used, the substance may be exposed to thecontinuous-operation light source before, after, or while exposing thesubstance to the pulsed light source.

In some embodiments, the pulsed light source may be switched OFF betweenpulses. In other embodiments, the pulsed light source may cycle betweena first state where it produces a high intensity output ofelectromagnetic radiation and a second state where it produces a lowintensity output of electromagnetic radiation, but the light source isnot switched OFF between pulses. In still other embodiments, a waveformof the pulses produced by the pulsed light source may be controlled,such that at least some of the pulses differ in pulse length or pulseintensity from other pulses.

In some embodiments, methods described herein may comprise: measuringviable microorganisms in a substance, identifying one or more types ofmicroorganisms in a substance, or any combination thereof using one ormore integrated computational elements; after measuring viablemicroorganisms or identifying one or more types of microorganisms in thesubstance, exposing the substance to a pulsed light source operable forforming rendering at least a portion of the microorganisms non-viable;and after or while exposing the substance to the pulsed light source,determining a microorganism load of the substance using one or moreintegrated computational elements.

The underlying theory behind using integrated computational elements forconducting analyses is described in more detail in the followingcommonly owned U.S. patents and patent application Publications, each ofwhich is incorporated herein by reference in its entirety: U.S. Pat.Nos. 6,198,531, 6,529,276, 7,123,844, 7,834,999, 7,911,605, 7,920,258,2009/0219538, 2009/0219539, and 2009/0073433. Accordingly, thetheoretical aspects of integrated computational elements will not bediscussed in any great detail herein.

FIG. 1 shows a schematic of an illustrative integrated computationalelement (ICE) 100. As illustrated in FIG. 1, ICE 100 may include aplurality of alternating layers 102 and 104 of varying thicknessesdisposed on optical substrate 106. In general, the materials forminglayers 102 and 104 have indices of refraction that differ (i.e., one hasa low index of refraction and the other has a high index of refraction),such as Si and SiO₂. Other suitable materials for layers 102 and 104 mayinclude, but are not limited to, niobia and niobium, germanium andgermania, MgF, and SiO. Additional pairs of materials having high andlow indices of refraction can be envisioned by one having ordinary skillin the art, and the composition of layers 102 and 104 is not consideredto be particularly limited. In some embodiments, the material withinlayers 102 and 104 can be doped, or two or more materials can becombined in a manner to achieve a desired optical response. In additionto solids, ICE 100 may also contain liquids (e.g., water) and/or gases,optionally in combination with solids, in order to produce a desiredoptical response. The material forming optical substrate 106 is notconsidered to be particularly limited and may comprise, for example,BK-7 optical glass, quartz, sapphire, silicon, germanium, zinc selenide,zinc sulfide, various polymers (e.g., polycarbonates,polymethylmethacrylate, polyvinylchloride, and the like), diamond,ceramics, and the like. Opposite to optical substrate 106, ICE 100 mayinclude layer 108 that is generally exposed to the environment of thedevice or installation in which it is used.

The number, thickness, and spacing of layers 102 and 104 may bedetermined using a variety of approximation methods based upon aconventional spectroscopic measurement of a sample. These methods mayinclude, for example, inverse Fourier transform (IFT) of the opticaltransmission spectrum and structuring ICE 100 as a physicalrepresentation of the IFT. The approximation methods convert the IFTinto a structure based on known materials with constant refractiveindices.

It should be understood that illustrative ICE 100 of FIG. 1 has beenpresented for purposes of illustration only. Thus, it is not impliedthat ICE 100 is predictive for any particular type of viable ornon-viable microorganism. Furthermore, it is to be understood thatlayers 102 and 104 are not necessarily drawn to scale and shouldtherefore not be considered as limiting of the present disclosure.Moreover, one having ordinary skill in the art will readily recognizethat the materials comprising layers 102 and 104 may vary depending onfactors such as, for example, the types of microorganisms being analyzedand the ability to accurately conduct their analysis, cost of goods,and/or chemical compatibility issues.

In addition, significant benefits can often be realized by combining theoutputs of two or more integrated computational elements with oneanother when analyzing a single constituent or characteristic ofinterest. Specifically, significantly increased detection accuracy maybe realized. Analysis techniques and devices utilizing combinations oftwo or more integrated computational elements are described in commonlyowned U.S. patent application Ser. Nos. 13/456,255, 13/456,264,13/456,283, 13/456,302, 13/456,327, 13/456,350, 13/456,379, 13/456,405,and 13/456,443, each filed on Apr. 26, 2012 and hereby incorporated byreference in their entireties.

To detect and analyze microorganisms using an integrated computationalelement, a substance containing the microorganisms may be illuminatedwith a source of electromagnetic radiation during or after its exposureto the pulsed light source. Suitable sources of electromagneticradiation may include, for example, infrared (including near-infrared)radiation, visible light, and/or ultraviolet radiation. In someembodiments, the substance may be stimulated to emit electromagneticradiation upon its illumination with the source of electromagneticradiation (e.g., fluorescent emission and/or phosphorescent emission).In some embodiments, the electromagnetic radiation may opticallyinteract with the substance and then optically interact with theintegrated computational element (e.g., via transmission, reflection,transflection, or through the use of evanescent radiation). In otherembodiments, the electromagnetic radiation may optically interact withthe integrated computational element and then optically interact withthe substance. In either instance, following optical interaction withthe integrated computational element and the substance, theelectromagnetic radiation may be received by a detector. The output ofthe detector may then be correlated with a property of the substance,such as the substance's microorganism load. As used herein, the term“optically interact” and variants thereof refer to the reflection,transmission, scattering, diffraction, or absorption of electromagneticradiation by a substance or an integrated computational element.

A wide variety of pulsed light sources may be suitable for use in theembodiments described herein. In some embodiments, the pulsed lightsource may produce electromagnetic radiation pulses of substantially asingle wavelength (i.e., a monochromatic or substantially monochromaticsource of electromagnetic radiation, which may also be coherent, such asa laser or light-emitting diode, for example). In other embodiments, thepulsed light source may be polychromatic and produce electromagneticradiation pulses exhibiting a range or plurality of wavelengths. In someembodiments, the pulsed light source may comprise a pulsed ultravioletlight source. In some embodiments, suitable pulsed light sources mayproduce at least a wavelength range of about 100 nm to about 280 nm. Asone of ordinary skill in the art will recognize, this wavelength rangeencompasses the ultraviolet C (UVC) range, which can be very effectivefor rendering microorganisms non-viable. Pulsed light sources producingother wavelength ranges may also be effective. In some embodiments,suitable pulsed light sources may produce at least a wavelength range ofabout 10 nm to about 100 nm, or about 280 nm to about 315 nm(ultraviolet B), or about 315 nm to about 400 nm (ultraviolet A), orabout 400 nm to about 700 nm (visible light). In some embodiments, abroad spectrum pulsed light source producing an output ofelectromagnetic radiation from about 100 nm to about 700 nm or fromabout 100 nm to about 800 nm may be used. Other suitable types ofelectromagnetic radiation that may be used in the pulsed light sourceinclude, for example, radio waves, microwave radiation, infrared andnear-infrared radiation, visible light, vacuum ultraviolet radiation,X-ray radiation and gamma ray radiation.

In some embodiments, the pulsed light source may comprise a pulsed xenonor pulsed krypton ultraviolet light source. That is, the pulsed lightsource may produce electromagnetic radiation generated by excitation ofthese gases. Pulsed xenon ultraviolet light, for example, may becharacterized as a polychromatic broad band emission having a wavelengthrange of about 180 nm to about 800 nm. As a non-limiting example, apulsed xenon ultraviolet light source may deliver energy to a substanceat a rate of about 10 Joules/second through emission of pulses having apower of approximately 1000 watts, a pulse length of about 10 ms, and afrequency of about 10 s⁻¹. For comparison, a continuous-operationmercury vapor ultraviolet light source also delivering energy to asubstance at a rate of about 10 Joules/second produces onlyapproximately a 10 watt continuous emission.

In some embodiments, the pulsed light source may comprise a mercuryvapor pulsed light source. As one of ordinary skill in the art willrecognize, mercury vapor ultraviolet light sources typically producemore discrete emission bands than do pulsed xenon or pulsed kryptonultraviolet light sources, although the latter ultraviolet light sourcesmay be more intense.

In addition to the wavelength range and type of electromagneticradiation, operational parameters that may be varied for the pulsedlight source include, for example, the light intensity (i.e., the energydensity), the pulse frequency, the pulse width, the pulse waveform andvariations thereof, the exposure time to a substance, or any combinationthereof. In some embodiments, a combination of short and long pulses maybe used, optionally varying the pulse intensity. In some or otherembodiments, a combination of short, medium, and long pulses or morecomplex pulse sequences may be used, optionally varying the pulseintensity.

In some embodiments, suitable pulsed light sources may produce lightintensities ranging between about 0.01 J/cm² and about 50 J/cm². In someembodiments, suitable pulsed light sources may produce light intensitiesranging between about 0.1 J/cm² and about 50 J/cm². Continuous-operationlight sources, in contrast, may not be amenable to ongoing operation atthe much higher light intensities that can be achieved with pulsed lightsources. In some embodiments, suitable pulsed light sources may producepulse frequencies ranging between about 1 s⁻¹ and about 10,000 s⁻¹ orbetween about 5 s⁻¹ and about 30 s⁻¹. In some embodiments, suitablepulsed light sources may produce pulse widths ranging between about 0.1ns and about 100 s, or between about 0.1 μs and about 100 μs, or betweenabout 1 ms and about 1 s. In some embodiments, suitable exposure timesof the pulsed light source to the substance may range between about 0.1s and about 60 minutes, or between about 1 s and about 15 minutes, orbetween about 10 s and about 10 minutes. In some embodiments, during theexposure time, sufficient energy may be supplied to the microorganismsto at least partially render them non-viable.

In some embodiments, determining a microorganism load of the substanceusing one or more integrated computational elements may comprisemeasuring viable microorganisms in the substance, measuring non-viablemicroorganisms in the substance, identifying one or more types ofmicroorganisms in the substance, or any combination thereof. Forexample, when analyzing bacteria, determining a microorganism load maycomprise measuring viable bacteria in the substance, measuringnon-viable bacteria in the substance, determining one or more types ofbacteria in the substance, or any combination thereof. In someembodiments, the methods described herein may comprise detecting aparticular subclass of microorganisms (e.g., aerobic and anaerobicbacteria) and/or how effective a pulsed light source has been inrendering them non-viable. In some embodiments, the methods may comprisedetecting a specific type (i.e., species) of microorganism and/or howeffective a pulsed light source has been in rendering it non-viable. Asone of ordinary skill in the art will recognize, some particular typesor species of microorganisms may be more problematic than others in agiven application, and their remediation may be more of a concern thanothers. For example, anaerobic bacteria may produce hydrogen sulfide asa metabolic product, which may be very undesirable for a number ofapplications, including subterranean operations, due to corrosion andtoxicity issues. Thus, the ability to rapidly determine the viability ofparticular types or species of microorganisms using the present methodsmay be particularly advantageous.

In some embodiments, determining a microorganism load of the substancemay take place in real-time or near real-time while the substance isbeing exposed to the pulsed light source. In other embodiments,determining a microorganism load of the substance may take place afterexposure to the pulsed light source takes place. That is, in someembodiments, determining a microorganism load of the substance may takeplace offline in non-real-time. In still other embodiments, the presentmethods may be used to follow a microorganism load of a substance withtime. For example, the kinetic growth or decline of a microorganismpopulation may be followed using one or more integrated computationalelements.

In some embodiments, the methods described herein may be used todetermine the microorganism load of a substance and the changes in themicroorganism load brought about through exposure to a pulsed lightsource. That is, in some embodiments, the present methods may be used todetermine the effectiveness of a pulsed light treatment for remediationof a microorganism contamination. In some embodiments, the methodsdescribed herein may be used to determine the microorganism load of thesubstance before and after its exposure to a pulsed light source. Forexample, in some embodiments, the methods may further comprise measuringthe viable microorganisms in the substance using an integratedcomputational element, before exposing the substance to the pulsed lightsource. Thus, in such embodiments, decreases in viable microorganismsfollowing exposure to a pulsed light source may be observed.

By evaluating the effectiveness of a pulsed light treatment uponmicroorganism levels, future treatments may be better designed toimprove their effectiveness and/or treatment of a substance may berepeated and optionally modified to reduce microorganism levels to asuitable level. In some embodiments, the methods described herein mayfurther comprise adjusting one or more operational parameters associatedwith the pulsed light source in response to the microorganism loaddetermined for the substance using the one or more integratedcomputational elements. Operational parameters of the pulsed lightsource that may be altered in response to the determined microorganismload may include, for example, the light intensity, the pulse width, thepulse frequency, the wavelength range, exposure time to the pulsed lightsource, the pulse waveform, the variety of pulse waveforms employed, thebias light intensity, or any combination thereof. As used herein, theterm “bias light intensity” refers to a low intensity state in a pulsedlight source that is not cycled completely to an OFF state followingdelivery of high intensity pulse of electromagnetic radiation. In someembodiments, the operational parameter(s) may be adjusted in real-timeor near real-time while measuring the microorganism load of thesubstance. In some embodiments, the present methods may further compriserepeating the exposure of the substance to the pulsed light sourcefollowing alteration of the pulsed light source's operationalparameter(s).

In still other embodiments, the present methods may further comprise useof a biocide in conjunction with the pulsed light source. That is, insome embodiments, the methods may further comprise introducing a biocideto the substance being treated with the pulsed light source. Biocidessuitable for use with particular microorganisms will be familiar to onehaving ordinary skill in the art. Use of a biocide in conjunction withthe pulsed light source may further improve the production of non-viablemicroorganisms. For example, the biocide may weaken the microorganismsand make them more susceptible to inactivation with the pulsed lightsource. In the alternative, a biocide may target a population ofmicroorganisms not targeted by the pulsed light treatment, or a biocidemay be more effective after the microorganisms are weakened by exposureto a pulsed light source. In some embodiments, the microorganisms may beexposed to the biocide prior to being exposed to the pulsed lightsource. In other embodiments, the microorganisms may be exposed to thebiocide while being exposed to the pulsed light source. In still otherembodiments, the microorganisms may be exposed to the biocide afterbeing exposed to the pulsed light source. The integrated computationalelement may be used to analyze the microorganisms at any point duringthis process.

In some embodiments, the methods described herein can further comprisedetermining a kill ratio for a population of microorganisms that hasbeen exposed to a pulsed light source. As used herein, the term “killratio” refers to the number of non-viable microorganisms present in asubstance after exposure to a pulsed light source relative to the numberof viable microorganisms present in the substance before exposure. Thekill ratio can be determined, in some embodiments, by quantifying theviable microorganisms before and after exposure to a pulsed light sourcetakes place. In other embodiments, non-viable microorganisms may bedetermined instead. In some embodiments, the kill ratio can be at leastabout 75%. In other embodiments, the kill ratio can be at least about80%, or at least about 85%, or at least about 90%, or at least about95%, or at least about 96%, or at least about 97%, or at least about98%, or at least about 99%. In some embodiments, if a desired kill ratiois not attained, the methods can further comprise repeating exposure tothe pulsed light source, as described above, changing one or moreoperational parameters of the pulsed light source and/orcontinuous-operation light source, if used, or performing a differentremediation treatment for controlling the microorganisms (e.g., abiocidal treatment).

The types of substances that may be treated using a pulsed light sourceand analyzed for microorganisms using the present methods are notbelieved to be particularly limited. In some embodiments, the substancemay comprise a fluid. In other embodiments, the substance may comprise asolid surface. In some embodiments, the fluid or solid surface may besubstantially opaque to visible light. As one of ordinary skill in theart will recognize, these types of substances may sometimes be lesseffectively treated with visible pulsed light treatments. However, bychoosing a wavelength or wavelength range of electromagnetic radiationwhere the fluid or solid surface is more transparent to theelectromagnetic radiation, an effective pulsed light treatment may stillbe realized. For example, oil, which is substantially opaque in thevisible region, may be substantially transparent to near-infraredelectromagnetic radiation. In addition, one having ordinary skill in theart will recognize that pulsed light sources may sometimes create shortterm transmission paths for electromagnetic radiation through asubstance through the creation of various excited electronic states.Thus, in some embodiments, a first pulse of electromagnetic radiationmay be used to create a substance in an excited electronic state that isthen transparent to a second pulse of electromagnetic radiation, inorder to effectively treat a substance with pulsed light. In some orother embodiments, a continuous-operation light source may be used toimprove the transparency of an opaque substance to electromagneticradiation in a manner similar to that described above.

As one of ordinary skill in the art will recognize, solid surfaces maybe particularly susceptible to growth of microorganisms thereon. One ofordinary skill in the art will further recognize that microorganismcontamination upon a surface may result in a number of deleteriouseffects including, for example, biofouling, permeability reduction,structural failure, corrosion, health hazards, and any combinationthereof. Contamination by microorganisms can be particularly problematicin a pipeline or like fluid conduit. In a pipeline or like fluidconduit, microorganisms can sometimes aggregate in joints, welds, seams,and the like, where they may significantly increase the risk ofstructural failure. As noted above, anaerobic bacteria may beparticularly problematic in this regard due to the hydrogen sulfide thatthey produce as a metabolic byproduct. The methods described herein maybe used to reduce the deleterious effects associated with microorganismcontamination. Solid surfaces that may be exposed to a pulsed lightsource and analyzed by the methods described herein include, forexample, pipeline surfaces, welds, proppant particulates, subterraneanformation surfaces, wellbore surfaces, medical device and surgicalinstrument surfaces, food preparation surfaces, reactor vessel surfaces,and the like.

In some embodiments, the fluid being exposed to a pulsed light sourceand analyzed by the present methods may comprise an aqueous fluid suchas water. In some or other embodiments, the fluid may comprise anoleaginous fluid, such as oil or a hydrocarbon. In some embodiments, thefluid may be static (i.e., not moving) while being analyzed by themethods described herein. In other embodiments, the fluid may be inmotion while being analyzed. In some embodiments, determining amicroorganism load of the substance may take place while the fluid isflowing (e.g., in a pipeline or like fluid conduit). In someembodiments, the fluid may be flowing while being exposed to the pulsedlight source.

Water used in subterranean operations can sometimes be obtained from anumber of “dirty” water sources, having varying levels of bacterial orother types of microorganism contamination therein. Althoughmicroorganism contamination may sometimes not be particularlyproblematic at ambient temperatures on the earth's surface, once thewater is introduced into a more favorable growth environment,microorganism levels and their detrimental effects may rapidly increase.For example, when introduced into a warm subterranean environment, evenlow levels of microorganisms can multiply quickly and potentially leadto damage of a subterranean formation. Likewise, favorable growthconditions may sometimes be found in a pipeline or like fluid conduit.In some cases, microorganisms may lead to biofouling of a subterraneansurface or pipeline surface. As discussed above, anaerobic bacteria maybe particularly detrimental when introduced into a subterraneanformation or a pipeline due to the hydrogen sulfide that they produce.In some cases, aerobic bacteria may be tolerable, at least to someextent. Rapidly multiplying microorganisms and their metabolicbyproducts can quickly clog and corrode production tubulars, plugformation fractures, and/or produce hydrogen sulfide which represents ahealth hazard and can lead to completion failure and loss of production.Accordingly, it can be highly desirable to reduce microorganism levelsbefore or while introducing a fluid to a subterranean formation.

In some embodiments, the methods described herein may further compriseintroducing a fluid into a subterranean formation, such as via awellbore.

In some embodiments, the fluid may comprise a treatment fluid or adrilling mud. As used herein, the term “treatment fluid” refers to afluid that is placed in a location (e.g., in a subterranean formation orin a pipeline or like fluid conduit) in order to perform a desiredfunction or to achieve a desired purpose. Treatment fluids can be usedin a variety of subterranean operations, including, but not limited to,drilling operations, production operations, stimulation operations,remediation operations, fluid diversion operations, secondary ortertiary enhanced oil recovery (EOR) operations, and the like. As usedherein, the terms “treat,” “treatment,” “treating,” and othergrammatical equivalents thereof refer to any operation that uses a fluidin conjunction with performing a desired function and/or achieving adesired purpose. The terms “treat,” “treatment,” and “treating,” as usedherein, do not imply any particular action by the fluid or anyparticular component thereof unless otherwise specified. Treatmentfluids can include, for example, fracturing fluids, acidizing fluids,conformance treatment fluids, damage control fluids, remediation fluids,scale removal and inhibition fluids, chemical floods, and the like. Insome embodiments, the treatment fluid or drilling mud may be exposed toa pulsed light source and analyzed using one or more integratedcomputational elements prior to being introduced to the subterraneanformation. In some or other embodiments, the treatment fluid or drillingmud may be exposed to a pulsed light source and analyzed using one ormore integrated computational elements while being introduced to thesubterranean formation. In still other embodiments, the treatment fluidor drilling mud may be exposed to the pulsed light source and analyzedusing one or more integrated computational elements while in thesubterranean formation. In some embodiments, the present methods may beused, at least in part, to render a produced fluid, such as a producedformation fluid or a produced treatment fluid, suitable for beingre-introduced to a subterranean formation.

In some embodiments, the methods described herein can further comprisedetermining suitable operational parameters for a pulsed light sourceused in conjunction with remediating microorganism contamination in asubstance. By determining the number and/or types of microorganismspresent in a substance before exposing the substance to a pulsed lightsource, more suitable operational parameters to address the particulartype and/or extent of microorganism contamination may be used. Forexample, in some embodiments, the exposure time of the substance to thepulsed light source may be changed and/or the pulse width, frequency,waveform, intensity and/or cycle may be altered in response toparticular quantities and/or types of microorganisms present in thesubstance. In some embodiments, determining suitable conditions for thepulsed light exposure may take place automatically under computercontrol. For example, a computer may select appropriate operationalparameters for the pulsed light source based upon input data of howeffectively substances having similar microorganism loads have beenremediated using a pulsed light source. In other embodiments,determining suitable operational parameters for the pulsed light sourcemay take place manually under the direction of an operator. In eithercase, determining suitable operational parameters for the pulsed lightsource may be impacted, at least in part, by the location in which thesubstance will be used. For example, if used under conditions not overlyconducive to microorganism growth, higher microorganism levels may besomewhat more tolerable in a substance. One of ordinary skill in the artwill recognize suitable levels of microorganisms that may ordinarily bepresent in a given application.

In some embodiments, the methods described herein may further comprisedetermining if the microorganism load of a fluid is suitable for beingintroduced to a subterranean formation. For example, if themicroorganism load of the fluid is unacceptably high, or if certaintypes of microorganisms are present in the fluid, the fluid may bedeemed unsuitable for subterranean introduction. Knowing themicroorganism load of the fluid and conditions present within a givensubterranean formation, one of ordinary skill in the art will be able todetermine a fluid's suitability for introduction to a given subterraneanformation. In various embodiments, determination of a fluid'ssuitability for introduction to a particular subterranean formation maybe made automatically under computer control or manually by an operator.In some embodiments, the methods may further comprise re-exposing thefluid to the pulsed light source, optionally after altering one or moreoperational parameters thereof, prior to or while the fluid is beingintroduced to the subterranean formation. In some or other embodiments,the fluid may be exposed to the pulsed light source after introductionto the subterranean formation.

In some embodiments, devices for detecting and analyzing microorganismsare described herein. In some embodiments, the devices may comprise apulsed light source configured to expose a substance to electromagneticradiation suitable for rendering one or more microorganisms non-viable;and one or more integrated computational elements configured fordetermining a microorganism load of the substance after or during itsexposure to the pulsed light source. In some embodiments, the pulsedlight source may comprise a pulsed ultraviolet light source. In someembodiments, the devices may further comprise a continuous-operationlight source, which may comprise a mercury vapor ultraviolet light, forexample. When used, the continuous-operation light source may illuminatea substance with electromagnetic radiation at the same and/or differenttime and/or position as the pulsed light source.

In some embodiments, the devices may be portable, such that they can beeasily transported to any substance needing remediation and/or analysisof a microorganism contamination thereon. In other embodiments, thedevices may be fixed in place, such as in a pipeline or tank, to provideongoing feedback of microorganism levels present therein. In someembodiments, the devices may be configured to analyze a staticsubstance. In other embodiments, the devices may be configured toanalyze a fluid substance that is in motion.

Therefore, the present invention is well adapted to attain the ends andadvantages mentioned as well as those that are inherent therein. Theparticular embodiments disclosed above are illustrative only, as thepresent invention may be modified and practiced in different butequivalent manners apparent to those skilled in the art having thebenefit of the teachings herein. Furthermore, no limitations areintended to the details of construction or design herein shown, otherthan as described in the claims below. It is therefore evident that theparticular illustrative embodiments disclosed above may be altered,combined, or modified and all such variations are considered within thescope and spirit of the present invention. The invention illustrativelydisclosed herein suitably may be practiced in the absence of any elementthat is not specifically disclosed herein and/or any optional elementdisclosed herein. While compositions and methods are described in termsof “comprising,” “containing,” or “including” various components orsteps, the compositions and methods can also “consist essentially of” or“consist of” the various components and steps. All numbers and rangesdisclosed above may vary by some amount. Whenever a numerical range witha lower limit and an upper limit is disclosed, any number and anyincluded range falling within the range is specifically disclosed. Inparticular, every range of values (of the form, “from about a to aboutb,” or, equivalently, “from approximately a to b,” or, equivalently,“from approximately a-b”) disclosed herein is to be understood to setforth every number and range encompassed within the broader range ofvalues. Also, the terms in the claims have their plain, ordinary meaningunless otherwise explicitly and clearly defined by the patentee.Moreover, the indefinite articles “a” or “an,” as used in the claims,are defined herein to mean one or more than one of the element that itintroduces. If there is any conflict in the usages of a word or term inthis specification and one or more patent or other documents that may beincorporated herein by reference, the definitions that are consistentwith this specification should be adopted. the invention claimed is:

1. A method comprising: providing a substance comprising a plurality ofviable microorganisms; exposing the substance to a light source for asufficient length of time to form at least some non-viablemicroorganisms; and determining a microorganism load of the substanceusing one or more integrated computational elements.
 2. The method ofclaim 1, wherein the light source comprises a pulsed light source. 3.The method of claim 2, wherein the pulsed light source comprises apulsed UV light source.
 4. The method of claim 2, wherein themicroorganism load of the substance is determined after or whileexposing the substance to the pulsed light source.
 5. The method ofclaim 4, wherein determining a microorganism load of the substance usingone or more integrated computational elements comprises measuring viablemicroorganisms in the substance, measuring non-viable microorganisms inthe substance, identifying one or more types of microorganisms in thesubstance, or any combination thereof.
 6. The method of claim 2, furthercomprising: before exposing the substance to the pulsed light source,measuring viable microorganisms in the substance, identifying one ormore types of microorganisms in the substance, or any combinationthereof using the one or more integrated computational elements.
 7. Themethod of claim 2, further comprising: introducing a biocide to thesubstance.
 8. The method of claim 2, further comprising: exposing thesubstance to a continuous-operation light source before, after, or whileexposing the substance to the pulsed light source.
 9. The method ofclaim 8, wherein the pulsed light source comprises a pulsed UV lightsource and the continuous-operation light source comprises a mercuryvapor UV light source.
 10. The method of claim 2, further comprising:adjusting one or more operational parameters associated with the pulsedlight source in response to the microorganism load determined for thesubstance.
 11. The method of claim 2, wherein the substance comprises afluid.
 12. The method of claim 11, wherein the fluid is flowing whiledetermining the microorganism load of the substance, exposing thesubstance to the pulsed light source, or both.
 13. The method of claim11, further comprising: introducing the fluid into a subterraneanformation.
 14. The method of claim 2, wherein the substance comprises asolid surface.
 15. The method of claim 14, wherein the solid surfacecomprises a fluid conduit.
 16. The method of claim 2, wherein thesubstance comprises a drinking water, a beverage, a foodstuff, aprocessing water, a waste water, a pharmaceutical, a cosmetic, a medicaldevice, an oil, a treatment fluid, a drilling mud, or any combinationthereof.
 17. The method of claim 2, wherein the microorganisms compriseat least one type of microorganism selected from the group consisting ofaerobic bacteria, anaerobic bacteria, protobacteria, protozoa,phytoplankton, viruses, fungi, alga, and any combination thereof.
 18. Amethod comprising: measuring viable microorganisms in a substance,identifying one or more types of microorganisms in a substance, or anycombination thereof using one or more integrated computational elements;after measuring viable microorganisms or identifying one or more typesof microorganisms in the substance, exposing the substance to a pulsedlight source operable for rendering at least a portion of themicroorganisms non-viable; and after or while exposing the substance tothe pulsed light source, determining a microorganism load of thesubstance using one or more integrated computational elements.
 19. Themethod of claim 18, wherein the microorganisms comprise bacteria. 20.The method of claim 19, wherein determining a microorganism load of thesubstance comprises measuring viable bacteria in the substance,measuring non-viable bacteria in the substance, identifying one or moretypes or species of bacteria in the substance, or any combinationthereof.
 21. The method of claim 18, further comprising: adjusting oneor more operational parameters associated with the pulsed light sourcein response to the microorganism load determined for the substance. 22.The method of claim 18, wherein the substance comprises a solid surface.23. The method of claim 18, wherein the substance comprises a fluid. 24.The method of claim 23, further comprising: introducing the fluid into asubterranean formation.
 25. The method of claim 18, further comprising:introducing a biocide to the substance.
 26. The method of claim 18,further comprising: exposing the substance to a continuous-operationlight source before, after, or while exposing the substance to thepulsed light source.
 27. A device comprising: a pulsed light sourceconfigured to expose a substance to electromagnetic radiation suitablefor rendering one or more microorganisms non-viable; and one or moreintegrated computational elements configured for determining amicroorganism load of the substance after or during its exposure to thepulsed light source.